Mutations in the genes for amyloid precursor protein (APP) and presenilins (PS1, PS2) increase production of ␤-amyloid 42 (A␤ 42 ) and cause familial Alzheimer's disease (FAD). Transgenic mice that express FAD mutant APP and PS1 overproduce A␤ 42 and exhibit amyloid plaque pathology similar to that found in AD, but most transgenic models develop plaques slowly. To accelerate plaque development and investigate the effects of very high cerebral A␤ 42 levels, we generated APP/PS1 double transgenic mice that coexpress five FAD mutations (5XFAD mice) and additively increase A␤ 42 production. 5XFAD mice generate A␤ 42 almost exclusively and rapidly accumulate massive cerebral A␤ 42 levels. Amyloid deposition (and gliosis) begins at 2 months and reaches a very large burden, especially in subiculum and deep cortical layers. Intraneuronal A␤ 42 accumulates in 5XFAD brain starting at 1.5 months of age (before plaques form), is aggregated (as determined by thioflavin S staining), and occurs within neuron soma and neurites. Some amyloid deposits originate within morphologically abnormal neuron soma that contain intraneuronal A␤. Synaptic markers synaptophysin, syntaxin, and postsynaptic density-95 decrease with age in 5XFAD brain, and large pyramidal neurons in cortical layer 5 and subiculum are lost. In addition, levels of the activation subunit of cyclin-dependent kinase 5, p25, are elevated significantly at 9 months in 5XFAD brain, although an upward trend is observed by 3 months of age, before significant neurodegeneration or neuron loss. Finally, 5XFAD mice have impaired memory in the Y-maze. Thus, 5XFAD mice rapidly recapitulate major features of AD amyloid pathology and may be useful models of intraneuronal A␤ 42 -induced neurodegeneration and amyloid plaque formation.

Summary Beta-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for β-amyloid (Aβ) production, is elevated in Alzheimer’s disease (AD). Here, we show that energy deprivation induces phosphorylation of the translation initiation factor eIF2α eIF2α-P), which increases the translation of BACE1. Salubrinal, an inhibitor of eIF2α-P phosphatase PP1c, directly increases BACE1 and elevates Aβ production in primary neurons. Preventing eIF2α phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominant negative eIF2α kinase PERK, or PERK inhibitor P58IPK blocks the energy deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increases levels of eIF2α-P, BACE1, Aβ, and amyloid plaques. Importantly, eIF2α-P and BACE1 are elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2α-P, and amyloid load are all correlated in humans with AD. These results strongly suggest that eIF2α phosphorylation increases BACE1 levels and causes Aβ overproduction, which could be an early, initiating molecular mechanism in sporadic AD.

The pathogenesis of Alzheimer's disease is highly complex. While several pathologies characterize this disease, amyloid plaques, composed of the β-amyloid peptide are hallmark neuropathological lesions in Alzheimer's disease brain. Indeed, a wealth of evidence suggests that β-amyloid is central to the pathophysiology of AD and is likely to play an early role in this intractable neurodegenerative disorder. The BACE1 enzyme is essential for the generation of β-amyloid. BACE1 knockout mice do not produce β-amyloid and are free from Alzheimer's associated pathologies including neuronal loss and certain memory deficits. The fact that BACE1 initiates the formation of β-amyloid, and the observation that BACE1 levels are elevated in this disease provide direct and compelling reasons to develop therapies directed at BACE1 inhibition thus reducing β-amyloid and its associated toxicities. However, new data indicates that complete abolishment of BACE1 may be associated with specific behavioral and physiological alterations. Recently a number of non-APP BACE1 substrates have been identified. It is plausible that failure to process certain BACE1 substrates may underlie some of the reported abnormalities in the BACE1-deficient mice. Here we review BACE1 biology, covering aspects ranging from the initial identification and characterization of this enzyme to recent data detailing the apparent dysregulation of BACE1 in Alzheimer's disease. We pay special attention to the putative function of BACE1 during healthy conditions and discuss in detail the relationship that exists between key risk factors for AD, such as vascular disease (and downstream cellular consequences), and the pathogenic alterations in BACE1 that are observed in the diseased state.

Mutations in the amyloid precursor protein (APP) cause early-onset Alzheimer's disease (AD), but the only genetic risk factor for late-onset AD is the varepsilon4 allele of apolipoprotein E (apoE), a major cholesterol carrier. Using Cre-lox conditional knockout mice, we demonstrate that lipoprotein receptor LRP1 expression regulates apoE and cholesterol levels within the CNS. We also found that deletion of APP and its homolog APLP2, or components of the gamma-secretase complex, significantly enhanced the expression and function of LRP1, which was reversed by forced expression of the APP intracellular domain (AICD). We further show that AICD, together with Fe65 and Tip60, interacts with the LRP1 promoter and suppresses its transcription. Together, our findings support that the gamma-secretase cleavage of APP plays a central role in regulating apoE and cholesterol metabolism in the CNS via LRP1 and establish a biological linkage between APP and apoE, the two major genetic determinants of AD.

Evidence suggests that β-amyloid (Aβ) peptide triggers a pathogenic cascade leading to neuronal loss in Alzheimer's disease (AD). However, the causal link between Aβ and neuron death in vivo remains unclear since most animal models fail to recapitulate the dramatic cell loss observed in AD. We have recently developed transgenic mice that overexpress human APP and PS1 with five familial AD mutations (5XFAD mice) and exhibit robust neuron death. Here, we demonstrate that genetic deletion of the β-secretase (BACE1) not only abrogates Aβ generation and blocks amyloid deposition but also prevents neuron loss found in the cerebral cortex and subiculum, brain regions manifesting the most severe amyloidosis in 5XFAD mice. Importantly, BACE1 gene deletion also rescues memory deficits in 5XFAD mice. Our findings provide strong evidence that Aβ ultimately is responsible for neuron death in AD and validate the therapeutic potential of BACE1-inhibiting approaches for the treatment of AD.

The use of statins, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that block the synthesis of mevalonate (and downstream products such as cholesterol and nonsterol isoprenoids), as a therapy for Alzheimer disease is currently the subject of intense debate. It has been reported that statins reduce the risk of developing the disorder, and a link between cholesterol and Alzheimer disease pathophysiology has been proposed. Moreover, experimental studies focusing on the cholesterol-dependent effects of statins have demonstrated a close association between cellular cholesterol levels and amyloid production. However, evidence suggests that statins are pleiotropic, and the potential cholesterol-independent effects of statins on amyloid precursor protein (APP) metabolism and amyloid ␤-peptide (A␤) genesis are unknown. In this study, we developed a novel in vitro system that enabled the discrete analysis of cholesterol-dependent and -independent (i.e. isoprenoid-dependent) statin effects on APP cleavage and A␤ formation. Given the recent interest in the role that intracellular A␤ may play in Alzheimer disease, we analyzed statin effects on both secreted and cell-associated A␤. As reported previously, low cellular cholesterol levels favored the ␣-secretase pathway and decreased A␤ secretion presumably within the endocytic pathway. In contrast, low isoprenoid levels resulted in the accumulation of APP, amyloidogenic fragments, and A␤ likely within biosynthetic compartments. Importantly, low cholesterol and low isoprenoid levels appeared to have completely independent effects on APP metabolism and A␤ formation. Although the implications of these effects for Alzheimer disease pathophysiology have yet to be investigated, to our knowledge, these results provide the first evidence that isoprenylation is involved in determining levels of intracellular A␤.A growing body of evidence suggests that the amyloid ␤-peptide (A␤) 1 plays a critical and early role in Alzheimer disease (AD) pathogenesis. AD is characterized by cerebral amyloid plaques, which are extracellular deposits of A␤ (1, 2). Overproduction of the 42-amino acid form of A␤ (A␤42) is associated with early onset familial AD, and A␤42 appears toxic to neurons in vitro and in vivo (reviewed in Refs. 3 and 4). Moreover, recent reports suggest that, in addition to extracellular A␤, the accumulation of intracellular A␤ may be involved in AD (5-15). Thus, much research has been devoted to understanding the role of A␤ in AD and to developing therapeutic strategies for reducing A␤ levels or toxicity. A␤ is cleaved from amyloid precursor protein (APP) by two proteases, the ␤-and ␥-secretases (reviewed in Refs. 4, 16, and 17). Initially, ␤-secretase (also known as BACE1) cuts APP at the N terminus of the A␤ domain to produce the membranebound APP C-terminal fragment (CTF) C99 and the secreted APP ectodomain APPs␤. C99 is the substrate of ␥-secretase, which cleaves to generate the C terminus of A␤. ␥-Secretase cleavage is heterogeneous and produces A␤ peptides of different le...

Amyloid plaques, composed of the amyloid ␤-protein (A␤), are hallmark neuropathological lesions in Alzheimer disease (AD) brain. A␤ fulfills a central role in AD pathogenesis, and reduction of A␤ levels should prove beneficial for AD treatment. A␤ generation is initiated by proteolysis of amyloid precursor protein (APP) by the ␤-secretase enzyme BACE1. Bace1 knockout (Bace1 ؊/؊ ) mice have validated BACE1 as the authentic ␤-secretase in vivo. BACE1 is essential for A␤ generation and represents a suitable drug target for AD therapy, especially because this enzyme is up-regulated in AD. However, although initial data indicated that Bace1 ؊/؊ mice lack an overt phenotype, the BACE1-mediated processing of APP and other substrates may be important for specific biological processes. In this minireview, topics range from the initial identification of BACE1 to the fundamental knowledge gaps that remain in our understanding of this protease. We address pertinent questions such as putative causes of BACE1 elevation in AD and discuss why, nine years since the identification of BACE1, treatments that address the underlying pathological mechanisms of AD are still lacking. Alzheimer Disease AD2 is the most common form of dementia, afflicting over 29 million people worldwide, a figure anticipated to rise exponentially within decades. Although the etiology remains enigmatic, AD appears to be brought about by both genetic and non-genetic factors. ϳ5% of AD cases are familial (FAD), caused by autosomal dominant mutations in either APP or the PS genes. The underlying cause(s) remain elusive for the majority of sporadic AD cases, although specific risk factors have been identified and include aging, the apolipoprotein E4 allele, certain vascular diseases, and TBI (1).Several major pathologies are observed in AD, and the amyloid hypothesis states that A␤ plays a critical early role, triggering a complex pathological cascade that leads to neurodegeneration (2). A strong genetic correlation exists between FAD and a neurotoxic form of fibrillogenic A␤, A␤42 (3). Pre-symptomatic FAD patients exhibit A␤42 elevations or elevations in A␤42 levels relative to levels of the less fibrillogenic peptide, A␤40, indicating that A␤42 may initiate pathophysiology. Patients with additional copies of the APP gene exhibit total A␤ overproduction and develop early-onset AD (1, 4). Data point toward a critical role for A␤42 in AD etiology, and strategies to lower brain A␤42 levels should be therapeutically beneficial in AD. A␤ GenerationA␤ is formed from endoproteolysis of APP, a type 1 membrane protein (Fig. 1) (5). BACE1 (␤-site APP-cleaving enzyme 1) is essential for initiating A␤ generation and cleaves the APP Asp ϩ1 residue to form the A␤ N terminus, APPs␤, and a C-terminal fragment, C99. BACE1 cleavage of APP is a prerequisite for ␥-secretase-mediated cleavage, and C99 is proteolyzed by ␥-secretase (a protein complex containing PS), which generates an AICD and A␤. This imprecise cleavage produces A␤ variants, including those ending at residues 40 ...

BackgroundThe β-secretase, BACE1, cleaves APP to initiate generation of the β-amyloid peptide, Aβ, that comprises amyloid plaques in Alzheimer’s disease (AD). Reducing BACE1 activity is an attractive therapeutic approach to AD, but complete inhibition of BACE1 could have mechanism-based side-effects as BACE1−/− mice show deficits in axon guidance, myelination, memory, and other neurological processes. Since BACE1+/− mice appear normal there is interest in determining whether 50% reduction in BACE1 is potentially effective in preventing or treating AD. APP transgenic mice heterozygous for BACE1 have decreased Aβ but the extent of reduction varies greatly from study to study. Here we assess the effects of 50% BACE1 reduction on the widely used 5XFAD mouse model of AD.Results50% BACE1 reduction reduces Aβ42, plaques, and BACE1-cleaved APP fragments in female, but not in male, 5XFAD/BACE1+/− mice. 5XFAD/BACE1+/+ females have higher levels of Aβ42 and steady-state transgenic APP than males, likely caused by an estrogen response element in the transgene Thy-1 promoter. We hypothesize that higher transgenic APP level in female 5XFAD mice causes BACE1 to no longer be in excess over APP so that 50% BACE1 reduction has a significant Aβ42 lowering effect. In contrast, the lower APP level in 5XFAD males allows BACE1 to be in excess over APP even at 50% BACE1 reduction, preventing lowering of Aβ42 in 5XFAD/BACE1+/− males. We also developed and validated a dot blot assay with an Aβ42-selective antibody as an accurate and cost-effective alternative to ELISA for measuring cerebral Aβ42 levels.Conclusions50% BACE1 reduction lowers Aβ42 in female 5XFAD mice only, potentially because BACE1 is not in excess over APP in 5XFAD females with higher transgene expression, while BACE1 is in excess over APP in 5XFAD males with lower transgene expression. Our results suggest that greater than 50% BACE1 inhibition might be necessary to significantly lower Aβ, given that BACE1 is likely to be in excess over APP in the human brain. Additionally, in experiments using the 5XFAD mouse model, or other Thy-1 promoter transgenic mice, equal numbers of male and female mice should be used, in order to avoid artifactual gender-related differences.